Optimization of Mariner Transposon for Bacillus

描述(申请人提供)：转座子是细菌遗传分析中最有用的工具之一。转座子不仅可以干扰操纵子或用可选择的标记标记有趣的等位基因，而且这些元件的特殊版本，携带复制或报告基因的起源，可以用来直接克隆或分析它们扰乱的操纵子的表达。对革兰氏阳性细菌的研究，包括炭疽芽孢杆菌，得益于在枯草杆菌中开发的转座子系统(Tn917)。虽然Tn917系统已在许多情况下成功使用，但它也不是没有局限性。最值得注意的是，在枯草杆菌的所有Tn917转位中，99%针对的是枯草杆菌染色体上的“热点”。最近，真核可转座元件Himar1已被修饰用于许多细菌系统，其中发生随机转座为二核苷酸序列(TA)。目前的申请寻求开发用于芽孢杆菌物种的Himar1。在芽孢杆菌中可选择的抗生素耐药盒将由转座子的反向末端重复序列(ITR)括起来，并放置在具有温度敏感的芽孢杆菌复制来源的质粒上。该质粒还将包括Himar1转座酶基因的一个高活性变体，该基因带有适合芽孢杆菌的表达元件。该系统将在基因上更易驯化的枯草杆菌中进行评估，一旦进行优化，就会用额外的元素(例如，报告基因系统、克隆的复制来源)进行修饰，并在炭疽杆菌中测试其有效性。在开发过程中，将使用为大肠杆菌开发的乳突试验的修改和芽孢杆菌特有的芽胞形成选择方案来寻找芽孢杆菌中高度活跃的Himar1转座酶变体。在这项研究中创造的转座子应该对芽孢杆菌和革兰氏阳性细菌都有用。此外，本研究中分离的高活性Himar1转座酶变体可以被整合到现有的Himar1系统中以提高其效率，并提供对转座酶活性或稳定性重要的元件的见解。

DESCRIPTION (provided by applicant): Transposable elements are among the most useful tools available for genetic analysis in bacteria. Not only can transposons disrupt operons or tag interesting alleles with selectable markers, but specialized versions of these elements, carrying origins of replication or reporter genes, can be used to directly clone or analyze the expression of the operons that they disrupt. Studies of Gram-positive bacteria, including Bacillus anthracis have benefited from a transposon system (Tn917) developed in B. subtilis. Although the Tn917 system has been successfully used in a number of instances, it is not without its limitations. Most notably, 99% of all Tn917 transpositions in B. subtilis target "hot spots" on the B. subtilis chromosome. Recently, the eukaryotic transposable element Himar1 has been modified for use in a number of bacterial systems, where random transposition occurs into a dinucleotide sequence (TA). The current application seeks to develop Himar1 for use in Bacillus species. Antibiotic resistance cassettes, selectable in Bacillus will be bracketed by the transposon's inverted terminal repeats (ITR) and placed on a plasmid with a temperature-sensitive Bacillus origin of replication. The plasmid will also include a hyperactive variant of the Himar1 transposase gene with expression elements appropriate for Bacillus. The system will be evaluated in the more genetically tractable B. subtilis, and once optimized, embellished with additional elements (e.g., a reporter gene system, origin of replication for cloning) and tested for effectiveness in B. anthracis. During development, Himar1 transposase variants, hyperactive in Bacillus will be sought using a modification of a papillation assay developed for use in Escherichia coli and a sporulation selection protocol unique to Bacillus. The transposons created in this study should be of use both in Bacillus species and Gram-positive bacteria in general. Additionally, the hyperactive Himar1 transposase variants isolated in this study could be incorporated into existing Himar1 systems to enhance their efficiency, and provide insights into elements of the transposase that are important for its activity or stability.